Project Summary/Abstract Hematopoietic stem cells (HSCs) are the functional units of bone marrow transplantation, which is used in the treatment of variety of blood cell diseases including leukemia and autoimmune disorders. Clinical use of HSCs is limited by the fact that they are rare cells, occurring at a frequency of only <1/20,000 bone marrow cells. Efforts to expand HSCs prior to transplant by ex vivo culturing have proven challenging and thus far such efforts have not translated to the clinic. Thus there is an important clinical need to find alternative strategies for either expanding the numbers of HSCs, or generating HSCs de novo. Numerous studies have shown that it is possible to experimentally reprogram the cellular identity of one cell type to another by enforced expression of transcription factors involved in the specification of the target cell type. The long-term objective of this project is to use this experimental paradigm and express transcription factors involved in specification of HSC fate and function in downstream blood cells to reprogram them back to a stem cell fate. In order to achieve this we must first identify the factors involved in specifying the fate and function of HSCs. This will be achieved using a large scale expression profiling strategy designed to allow us to identify transcription factors uniquely expressed in the stem cell compartment compared to the other cells of the blood system. Our preliminary studies have identified 28 such genes. Once identified we will then clone these factors into a vector delivery system to allow us to express combinations of these genes in downstream blood cells. We will then test whether or not downstream blood cells receiving such factors are able of function like HSCs by using a wide variety of stem cells assays. Once reprogramming has been achieved, it will then be important to determine the minimal combination of factors able to mediate reprogramming. During the last phase of the project we will attempt to elucidate the molecular mechanisms underlying HSC reprogramming. Successful identification of factors capable of reprogramming committed blood cells to a HSC fate has the potential to advance our basic understanding of hematopoietic stem cell biology, and contribute to their clinical utility in a number of ways. For example, identification of factors capable of reprogramming committed blood cells to HSCs will provide important insights the molecular mechanisms involved in specifying the fundamental stem cell properties of self-renewal and multi-potency. In terms of clinical application, if successful, our approach opens the possibility of generating patient HSCs in great numbers for use in bone marrow transplantation, or for the study of blood disorders.